Akiyoshi Moriwaki

Oxytocin improves long-lasting spatial memory during motherhood through MAP kinase cascade

Oxytocin is an essential hormone for mammalian labor and lactation. Here, we show a new function of oxytocin in causing plastic changes in hippocampal synapses during motherhood. In oxytocin-perfused hippocampal slices, one-train tetanus stimulation induced long-lasting, long-term potentiation (L-LTP) and phosphorylation of cyclic AMP–responsive element binding protein (CREB), and MAP kinase inhibitors blocked these inductions. An increase in CREB phosphorylation and L-LTP induced by one-train tetanus were observed in the multiparous mouse hippocampus without oxytocin application. Furthermore, intracerebroventricular injection of oxytocin in virgin mice improved long-term spatial learning in vivo, whereas an injection of oxytocin antagonist in multiparous mice significantly inhibited the improved spatial memory, L-LTP and CREB phosphorylation. These findings indicate that oxytocin is critically involved in improving hippocampus-dependent learning and memory during motherhood in mice.

A new cell-permeable peptide allows successful allogeneic islet transplantation in mice

Calcineurin inhibitors such as cyclosporine A and FK506 have been used for transplant therapy and treatment of autoimmune diseases. However, the inhibition of calcineurin outside the immune system has a number of side effects, including hyperglycemia. In the search for safer drugs, we developed a cell-permeable inhibitor of NFAT (nuclear factor of activated T cells) using the polyarginine peptide delivery system. This peptide provided immunosuppression for fully mismatched islet allografts in mice. In addition, it did not affect insulin secretion, whereas FK506 caused a dose-dependent decrease in insulin secretion. Cell-permeable peptides can thus provide a new strategy for drug development and may eventually be useful clinically.

Cdk5/p35 Regulates Neurotransmitter Release through Phosphorylation and Downregulation of P/Q-Type Voltage-Dependent Calcium Channel Activity

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase with close structural homology to the mitotic Cdks. The complex of Cdk5 and p35, the neuron-specific regulatory subunit of Cdk5, plays important roles in brain development, such as neuronal migration and neurite outgrowth. Moreover, Cdk5 is thought to be involved in the promotion of neurodegeneration in Alzheimers disease. Cdk5 is abundant in mature neurons; however, its physiological functions in the adult brain are unknown. Here we show that Cdk5/p35 regulates neurotransmitter release in the presynaptic terminal. Both Cdk5 and p35 were abundant in the synaptosomes. Roscovitine, a specific inhibitor of Cdk5 in neurons, induced neurotransmitter release from the synaptosomes in response to membrane depolarization and enhanced the EPSP slopes in rat hippocampal slices. The electrophysiological study using each specific inhibitor of the voltage-dependent calcium channels (VDCCs) and calcium imaging revealed that roscovitine enhanced Ca2+ influx from the P/Q-type VDCC. Moreover, Cdk5/p25 phosphorylated the intracellular loop connecting domains II and III (LII-III) between amino acid residues 724 and 981 of isoforms cloned from rat brain of the α1A subunit of P/Q-type Ca2+ channels. The phosphorylation inhibited the interaction of LII-IIIwith SNAP-25 and synaptotagmin I, which were plasma membrane solubleN-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins and were required for efficient neurotransmitter release. These results strongly suggest that Cdk5/p35 inhibits neurotransmitter release through the phosphorylation of P/Q-type VDCC and downregulation of the channel activity.

Increase in the calcium level following anodal polarization in the rat brain

The accumulation of calcium ions (Ca) was examined in the rat brain by means of 45Ca autoradiography following the application of a weak anodal direct current to the surface of the sensorimotor cortex. Repetition of the anodal polarization with 3.0 microA for 30 min caused more Ca to accumulate in the cerebral cortex. The degree and extent of accumulation was greater in the hemisphere ipsilateral to the polarization than in the other. Accumulation was also noted in the hippocampus and thalamus. Ca accumulation was detected after 24 h and it remained virtually constant up to 72 h after the last polarization. The results suggest that a long-lasting disturbance of Ca homeostasis is involved in the cortical plastic changes seen following anodal polarization.

Dopamine transporter cysteine mutants: second extracellular loop cysteines are required for transporter expression.

Abstract: Studies with thiol‐modifying reagents have suggested that cysteines might play important roles in the function of the dopamine transporter (DAT). To identify DAT cysteines with important thiol groups, we have studied six mutant dopamine transporters in which cysteines were replaced by alanines. Substitutions of cysteines assigned to the DATs second putative extracellular loop—positions 180 and 189—dramatically decreased the expression of the mutant transporters. Substitutions at positions 90, 242, 305, and 345 had no significant effect in decreasing dopamine uptake, MPP+ uptake, or cocaine analogue binding. Immunostaining COS cells transfected with Cys180 and Cys189 to Ala mutants revealed reduced membrane staining and prominent staining in perinuclear regions consistent with Golgi apparatus. These results suggest that cysteines in the DAT second extracellular loop may provide sulfide residues crucial to full transporter expression, at least in part, through interference with membrane insertion. Conceivably, they might also provide the targets for the influences of thiol‐modifying reagents in modifying the function of the wild‐type DAT expressed in striatal membranes.

Biphasic effects of polarizing current on adenosine-sensitive generation of cyclic AMP in rat cerebral cortex

Cyclic AMP accumulation elicited by adenosine was investigated in cortical slices of rats following the application of a weak anodal direct current (anodal polarization) to the unilateral sensorimotor cortex. Anodal polarization with 3.0 microA for 30 min caused an increase in the adenosine-elicited accumulation of cyclic AMP in the polarized cortex, in which case the increase in the polarized cortical region was highlighted by repeated applications of the currents. Polarization with 0.3 microA for 30 min decreased the cyclic AMP accumulation, and polarization with 30.0 microA for 30 min had no effect. When applied for 3 h, the polarizing currents at all of the intensities tested decreased the cyclic AMP accumulation. The results indicate that anodal polarization has biphasic effects on adenosine-elicited accumulation of cyclic AMP in the cortex. Alterations in the cyclic AMP generation are suggested to form the neurochemical basis of central and behavioral activity induced by anodal polarization.

Enhanced synaptic transmission and reduced threshold for LTP induction in fyn‐transgenic mice

To elucidate the physiological role of Fyn, we analysed the properties of synaptic transmission and synaptic plasticity in hippocampal slices of mice overexpressing either wild‐type Fyn (w‐Fyn) or its constitutively active mutant (m‐Fyn). These fyn‐transgenes were driven by the calcium/calmodulin‐dependent protein kinase IIα promoter which turned on in the forebrain neurons including hippocampal pyramidal cells and in late neural development. In the hippocampal slices expressing m‐Fyn the paired‐pulse facilitation was reduced and the basal synaptic transmission was enhanced. A weak theta‐burst stimulation, which was subthreshold for the induction of long‐term potentiation (LTP) in control slices, elicited LTP in CA1 region of the slices expressing m‐Fyn. When a relatively strong stimulation was applied, the magnitude of LTP in m‐Fyn slices was similar to that in control slices. By contrast, the basal synaptic transmission and the threshold for the induction of LTP were not altered in the slices overexpressing wild‐type Fyn. To examine the effect of expression of m‐Fyn on GABAergic inhibitory system, we applied bicuculline, a GABAA receptor blocker, to the hippocampal slices. The ability of bicuculline to enhance excitatory postsynaptic potentials was attenuated in slices expressing m‐Fyn, suggesting that the overexpression of m‐Fyn reduced the GABAergic inhibition. The enhancement of synaptic transmission and the reduction of GABAergic inhibition may contribute to the enhanced seizure susceptibility in the mice expressing m‐Fyn. Thus, these results suggest that regulation of Fyn tyrosine kinase activity is important for both synaptic transmission and plasticity.

Calcineurin inhibitors, FK506 and cyclosporin A, suppress the NMDA receptor-mediated potentials and LTP, but not depotentiation in the rat hippocampus

The effects of FK506, a Ca2+/calmodulin-dependent phosphatase 2B (calcineurin) inhibitor, on the NMDA receptor-mediated potentials and synaptic plasticity were investigated in the CA1 region of the rat hippocampus. Bath application of FK506 (50 microM) produced a 45% inhibition on the NMDA receptor-mediated potentials. FK506 also inhibited the induction of long-term potentiation (LTP), but had no effect on the depotentiation in the CA1 hippocampus. Cyclosporin A (100 microM), another calcineurin inhibitor, mimicked the effects of FK506 on the NMDA responses and synaptic plasticity. These results suggest that FK506 inhibits the activity of NMDA receptors via the involvement of calcineurin. The differential effects of FK506 on LTP and depotentiation may attribute to the partial inhibition on the activity of NMDA receptors and the subsequent attenuation of intracellular Ca2+ increase.

Changes in the expression of novel Cdk5 activator messenger RNA (p39nck5ai mRNA) during rat brain development.

We previously reported that a neuron-specific Cdk5 activator, p35nck5ai, was most prominent in the newborn rat brain. In the adult brain, the expression decreased in most regions except hippocampus and primary olfactory cortex. A novel neuron-specific Cdk5 activator, p39nck5ai, has been recently cloned. To clarify whether two activators were differentially distributed throughout brain development, in this study, we examined the spatial and temporal expression of p39nck5ai in the development rat brain. Northern blot analysis showed that p39nck5ai expression was low in 15-day old fetuses and newborn, and was most prominent in the 1-3 week-old rat brains. In the adult rat brain, expression declined to the same level as in newborn rat brain. In situ hybridization showed that p39nck5ai mRNA was weakly expressed in all neurons of all regions in the newborn rat brain and the transcriptional level was highest in all regions in the 3 week-old rat brain. In the adult, expression was decreased in most neurons except Purkinje and granule cells in the cerebellum which retained high levels. These results suggest that p35nck5a and p39nck5ai may have different functional roles in distinct brain regions during different states of the rat brain development.

HIV-1 Inhibits Long-Term Potentiation and Attenuates Spatial Learning

Although memory deficits have been clearly documented in patients with human immunodeficiency virus type‐1 (HIV‐1) infection, the physiological basis of this dysfunction is poorly understood. We focused on Tat, a viral protein released from HIV‐1–infected cells and investigated its effect on spatial learning in adult mice. An intracerebroventricular injection of Tat leads to attenuation of spatial learning accompanied by suppression of long‐term potentiation (LTP), the cellular basis of spatial learning, in hippocampal cornu ammonis 1 pyramidal neurons. Tat facilitates extrasynaptic but not synaptic N‐methyl‐D‐aspartate (NMDA) receptor activity. Taken together, these data provide strong evidence that the Tat pathway underlies the development of memory dysfunction in patients with HIV‐1 infection and suggest a causal relationship between Tat, the facilitation of extrasynaptic NMDA receptor activity, inhibition of LTP, and attenuation of spatial learning.